Antimicrobial resistance characteristics and fitness of Gram-negative fecal bacteria from volunteers treated with minocycline or amoxicillin.

A yearlong study was performed to examine the effect of antibiotic administration on the bacterial gut flora. Gram-negative facultative anaerobic bacteria were recovered from the feces of healthy adult volunteers administered amoxicillin, minocycline or placebo, and changes determined in antimicrobial resistance (AMR) gene carriage. Seventy percent of the 1039 facultative anaerobic isolates recovered were identified by MALDI-TOF as Escherichia coli. A microarray used to determine virulence and resistance gene carriage demonstrated that AMR genes were widespread in all administration groups, with the most common resistance genes being bla TEM, dfr, strB, tet(A), and tet(B). Following amoxicillin administration, an increase in the proportion of amoxicillin resistant E. coli and a three-fold increase in the levels of bla TEM gene carriage was observed, an effect not observed in the other two treatment groups. Detection of virulence genes, including stx1A, indicated not all E. coli were innocuous commensals. Approximately 150 E. coli collected from 6 participants were selected for pulse field gel electrophoresis (PFGE), and a subset used for characterisation of plasmids and Phenotypic Microarrays (PM). PFGE indicated some E. coli clones had persisted in volunteers for up to 1 year, while others were transient. Although there were no unique characteristics associated with plasmids from persistent or transient isolates, PM assays showed transient isolates had greater adaptability to a range of antiseptic biocides and tetracycline; characteristics which were lost in some, but not all persistent isolates. This study indicates healthy individuals carry bacteria harboring resistance to a variety of antibiotics and biocides in their intestinal tract. Antibiotic administration can have a temporary effect of selecting bacteria, showing co-resistance to multiple antibiotics, some of which can persist within the gut for up to 1 year

Impact of three ampicillin dosage regimens on selection of ampicillin resistance in Enterobacteriaceae and excretion of blaTEM genes in swine feces

The aim of this study was to assess the impact of three ampicillin dosage regimens on ampicillin resistance among Enterobacteriaceae recovered from swine feces using phenotypic and genotypic approaches. Phenotypically, ampicillin resistance was determined from the percentage of resistant Enterobacteriaceae and MICs of E. coli isolates. The pool of ampicillin resistance genes was also monitored by quantification of blaTEM genes, which code for the most frequently produced β-lactamases in Gram-negative bacteria, using a newly-developed real-time PCR assay. Ampicillin was administered intramuscularly and by oral route to fed or fasted pigs for 7 days at 20 mg/kg. The average percentage of resistant Enterobacteriaceae before treatment was between 2.5% and 12% and blaTEM genes quantities were below 107 copies/g of feces. By days four and seven, the percentage of resistant Enterobacteriaceae exceeded 50% in all treated groups, with some highly resistant strains (MIC>256µg/mL). In the control group, blaTEM genes quantities fluctuated between 104 - 106 copies/g of feces, whereas they fluctuated between 106-108 and 107-109 copies/g of feces for intramuscular and oral routes, respectively. Whereas phenotypic evaluations did not discriminate between the three ampicillin dosage regimens, blaTEM genes quantification was able to differentiate between the effects of two routes of ampicillin administration. Our results suggest that fecal blaTEM genes quantification provides a sensitive tool to evaluate the impact of ampicillin administration on the selection of ampicillin resistance in the digestive microflora and its dissemination in the environment

Detection of extended spectrum B-lactamases in urinary isolates of Klebsiella pneumoniae in relation to Bla SHV, Bla TEM and Bla CTX-M gene carriage

Background: Resistance to contemporary broad-spectrum β-lactam antibiotics mediated by extended-spectrum β-lactamases (ESBLs) is increasing worldwide. Klebsiella pneumoniae, an important cause of nosocomial and community acquired urinary tract infections has rapidly become the most common ESBL producing organism. We examined ESBL production in urinary isolates of K. pneumoniae in relation to the presence of bla SHV, bla TEM and bla CTX-M genes. Methods: Antibiotic resistance of 51 clinical isolates of K. pneumoniae was determined to amoxicillin, amikacin, ceftazidime, cefotaxime, cefteriaxon, ceftizoxime, gentamicin, ciprofloxacin and nitrofurantoin by disc diffusion. Minimum inhibitory concentrations were also measured for ceftazidime, cefotaxime, cefteriaxon, ceftizoxime and ciprofloxacin. ESBL production was detected by the double disc synergy test and finally, presence of the bla SHV, bla TEM and bla CTX-M genes were shown using specific primers and PCR. Results: Disc diffusion results showed that 96.08 % of the isolates were resistant to amoxicillin followed by 78.43 % resistance to nitrofurantoin, 49.02 % to amikacin and ceftazidime, 41.17 % to ceftriaxone, 37.25% resistance to cefotaxime and ceftizoxime, and 29.42 % to gentamicin and ciprofloxacin. Both resistant and intermediately resistant organisms were resistant in MIC determinations. Twenty two isolates (43.14%) carried bla SHV, 18 (35.29%) had bla TEM and 16 (31.37%) harbored bla CTX-M genes. ESBL production was present in 14 isolates (27.45 %) of which, 3 did not harbor any of the 3 genes. Among the non- ESBL producers, 9 lacked all 3 genes and 2 carried them all. Conclusion: No relation was found between gene presence and ESBL expression

A Population-Based Surveillance Study of Shared Genotypes of Escherichia coli Isolates from Retail Meat and Suspected Cases of Urinary Tract Infections.

There is increasing evidence that retail food may serve as a source of Escherichia coli that causes community-acquired urinary tract infections, but the impact of this source in a community is not known. We conducted a prospective, population-based study in one community to examine the frequency of recovery of uropathogenic E. coli genotypes from retail meat samples. We analyzed E. coli isolates from consecutively collected urine samples of patients suspected to have urinary tract infections (UTIs) at a university-affiliated health service and retail meat samples from the same geographic region. We genotyped all E. coli isolates by multilocus sequence typing (MLST) and tested them for antimicrobial susceptibility. From 2016 to 2017, we cultured 233 E. coli isolates from 230 (21%) of 1,087 urine samples and 177 E. coli isolates from 120 (28%) of 427 retail meat samples. Urine samples contained 61 sequence types (STs), and meat samples had 95 STs; 12 STs (ST10, ST38, ST69, ST80, ST88, ST101, ST117, ST131, ST569, ST906, ST1844, and ST2562) were common to both. Thirty-five (81%) of 43 meat isolates among the 12 STs were from poultry. Among 94 isolates in the 12 STs, 26 (60%) of 43 retail meat isolates and 15 (29%) of 51 human isolates were pan-susceptible (P < 0.005). We found that 21% of E. coli isolates from suspected cases of UTIs belonged to STs found in poultry. Poultry may serve as a possible reservoir of uropathogenic E. coli (UPEC). Additional studies are needed to demonstrate transmission pathways of these UPEC genotypes and their food sources.IMPORTANCE Community-acquired urinary tract infection caused by Escherichia coli is one of the most common infectious diseases in the United States, affecting approximately seven million women and costing approximately 11.6 billion dollars annually. In addition, antibiotic resistance among E. coli bacteria causing urinary tract infection continues to increase, which greatly complicates treatment. Identifying sources of uropathogenic E. coli and implementing prevention measures are essential. However, the reservoirs of uropathogenic E. coli have not been well defined. This study demonstrated that poultry sold in retail stores may serve as one possible source of uropathogenic E. coli This finding adds to a growing body of evidence that suggests that urinary tract infection may be a food-borne disease. More research in this area can lead to the development of preventive strategies to control this common and costly infectious disease

Seasonal variations in antibiotic resistance gene transport in the Almendares River, Havana, Cuba

Numerous studies have quantified antibiotic resistance genes (ARG) in rivers and streams around the world, and significant relationships have been shown that relate different pollutant outputs and increased local ARG levels. However, most studies have not considered ambient flow conditions, which can vary dramatically especially in tropical countries. Here, ARG were quantified in water column and sediment samples during the dry- and wet-seasons to assess how seasonal and other factors influence ARG transport down the Almendares River (Havana, Cuba). Eight locations were sampled and stream flow estimated during both seasons; qPCR was used to quantify four tetracycline, two erythromycin, and three beta-lactam resistance genes. ARG concentrations were higher in wet-season versus dry-season samples, which combined with higher flows, indicated much greater ARG transport downstream during the wet-season. However, water column ARG levels were more spatially variable in the dry-season than the wet-season, with the proximity of waste outfalls strongly influencing local ARG levels. Results confirm that dry-season sampling provides a useful picture of the impact of individual waste inputs on local stream ARG levels, whereas the majority of ARGs in this tropical river were transported downstream during the wet-season, possibly due to re-entrainment of ARG from sediments

Analysis for prevalence and physical linkages amongst integrons, ISEcp1, ISCR1, Tn21 and Tn7 encountered in Escherichia coli strains from hospitalized and non-hospitalized patients in Kenya during a 19-year period (1992-2011)

Background: We determined the prevalence and evidence for physical linkage amongst integrons, insertion sequences, Tn21 and Tn7 transposons in a collection of 1327 E. coli obtained over a 19-year period from patients in Kenya. Results: The prevalence of class 1 integrons was 35%, class 2 integrons were detected in 3 isolates but no isolate contained a class 3 integron. Integron lacking the 3'-CS or those linked to sul3 gene or IS26 or those containing the ISCR1 were only detected in multidrug resistant (MDR) strains. The dfrAs were the most common cassettes and their prevalence was: -dfrA1( 28%), dfrA12( 20%), dfA17( 9%), dfrA7( 9%), and dfrA16( 5%). The aadA were the second most abundant cassettes and their prevalence was: -aadA1( 25%), aadA2( 21%), and aadA5( 14%). Other cassettes occurred in lower prevalence of below 5%. Prevalence of Tn21, ISEcp1, ISCR1 and IS26 was 22%, 10%, 15%, and 7% respectively. Majority of Tn21 containing integrons carried a complete set of transposition genes while class 2 integrons were borne on Tn7 transposon. The qnrA genes were detected in 34( 3%) isolates while 19( 1%) carried qnrB. All qnr genes were in MDR strains carrying integrons containing the ISCR1. Close to 88% of blaTEM-52 were linked to IS26 while = 80% of blaCTX-Ms and blaCMYs were linked to ISEcp1. Only a few studies have identified a blaCTX-M-9 containing an ISEcp1 element as reported in this study. Multiple genetic elements, especially those borne on incIl, incFII, and incL/M plasmids, and their associated resistance genes were transferrable en bloc to E. coli strain J53 in mating experiments. Conclusions: This is the first detailed study on the prevalence of selected elements implicated in evolution of resistance determinants in a large collection of clinical E. coli in Africa. Proliferation of such strains carrying multiple resistance elements is likely to compromise the use of affordable and available treatment options for majority of poor patients in Africa. There is therefore a need to monitor the spread of these highly resistant strains in developing countries through proper infection control and appropriate use of antimicrobials

Designing antibiotic cycling strategies by determining and understanding local adaptive landscapes

The evolution of antibiotic resistance among bacteria threatens our continued ability to treat infectious diseases. The need for sustainable strategies to cure bacterial infections has never been greater. So far, all attempts to restore susceptibility after resistance has arisen have been unsuccessful, including restrictions on prescribing [1] and antibiotic cycling [2,3]. Part of the problem may be that those efforts have implemented different classes of unrelated antibiotics, and relied on removal of resistance by random loss of resistance genes from bacterial populations (drift). Here, we show that alternating structurally similar antibiotics can restore susceptibility to antibiotics after resistance has evolved. We found that the resistance phenotypes conferred by variant alleles of the resistance gene encoding the TEM {\beta}-lactamase (blaTEM) varied greatly among 15 different {\beta}-lactam antibiotics. We captured those differences by characterizing complete adaptive landscapes for the resistance alleles blaTEM-50 and blaTEM-85, each of which differs from its ancestor blaTEM-1 by four mutations. We identified pathways through those landscapes where selection for increased resistance moved in a repeating cycle among a limited set of alleles as antibiotics were alternated. Our results showed that susceptibility to antibiotics can be sustainably renewed by cycling structurally similar antibiotics. We anticipate that these results may provide a conceptual framework for managing antibiotic resistance. This approach may also guide sustainable cycling of the drugs used to treat malaria and HIV

The effect of combined sewer overflows on the abundance of antibiotic resistance genes and bacteria in the James River

Antibiotic resistance is a major threat to human health. Clinical situations are the main focus for antibiotic resistance research, but understanding the spread of resistance in the environment is also vital. A major contributor to this spread is wastewater from combined sewer overflow (CSO) events. The effect of CSO events on antibiotic resistance in the James River near Richmond, Virginia was studied using genomic and microbiological approaches. The abundance of genes associated with resistance to quinolones (qnrA) and tetracycline (tetW) was strongly correlated with the presence of fecal indicator bacteria (E. coli abundance) as well as total nitrogen and phosphorus loads, which suggests an anthropogenic source of these genes. Abundance of the blaTEM gene, which confers resistance to β-lactam antibiotics, was elevated during CSO events and increased with precipitation and river discharge. Bacteria isolated during a CSO event were resistant to more antibiotics and had higher multi-drug resistance when compared to isolates from a non-event. This study demonstrated that CSO events are contributing to the spread of antibiotic resistance

Serotype epidemiology and multidrug resistance patterns of Salmonella enterica infecting humans in Italy

BACKGROUND: Salmonella enterica is the zoonotic agent most frequently responsible for foodborne infections in humans worldwide. In this work the presence of S. enterica was investigated in 734 unique enteropathogenic isolates collected from human patients between 2011 and 2012. RESULTS: All Salmonella spp. isolates were subjected to serotyping and antimicrobial susceptibility testing. Isolates displaying phenotypes and antimicrobial susceptibility profiles different from the reference strains were genotipically characterized. Several plasmid-embedded resistance determinants were identified and characterized. Non-typhoidal serotypes were most frequently diagnosed; monophasic Salmonella typhimurium 1,4 [5],12:i- and S. typhimurium represented the most prevalent serovars. Five isolates displayed phenotypes with extremely reduced susceptibility to antimicrobials: we detected multidrug resistance elements belonging to Ambler class A and class C in two non-typhoidal S. enterica serovars, i.e. Rissen and monophasic S. typhimurium 1,4 [5],12:i-, and in one typhoidal serovar, i.e., Paratyphi B. These resistance determinants have been so far almost exclusively associated with non-Salmonella members of the Enterobacteriaceae family. Alarmingly, two colistin resistant Salmonella enteritidis were also found. CONCLUSIONS: This work draws the attention to the still low, but rising, percentage of multidrug resistant Salmonella isolates infecting humans in Italy. Our results suggest that prompt monitoring of Salmonella serovar dissemination and resistance to antimicrobials is highly required

Broad-spectrum β-lactamases among Enterobacteriaceae of animal origin: molecular aspects, mobility and impact on public health

Broad-spectrum β-lactamase genes (coding for extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases) have been frequently demonstrated in the microbiota of food-producing animals. This may pose a human health hazard since these genes may be present in zoonotic bacteria, which would cause a direct problem. They can also be present in commensals, which may act as a reservoir of resistance genes for pathogens causing disease both in humans and animals. Broad-spectrum β-lactamase genes are frequently located on mobile genetic elements, such as plasmids, transposons and integrons, which often also carry additional resistance genes. This could limit treatment options for infections caused by broad-spectrum β-lactam-resistant microorganisms. This review addresses the growing burden of broad-spectrum β-lactam resistance among Enterobacteriaceae isolated from food, companion and wild animals worldwide. To explore the human health hazard, the diversity of broad-spectrum β-lactamases among Enterobacteriaceae derived from animals is compared with respect to their presence in human bacteria. Furthermore, the possibilities of the exchange of genes encoding broad-spectrum β-lactamases – including the exchange of the transposons and plasmids that serve as vehicles for these genes – between different ecosystems (human and animal) are discussed